Production of itaconic acid

ABSTRACT

The invention relates to a nucleic acid sequence encoding an itaconate transporting Major Facilitator Superfamily Transporter (MFST) gene sequence and the protein encoded thereby. Preferably said sequence is the nucleic acid that comprises the sequence of ATEG_09972.1 of  Aspergillus terreus  or homologues thereof. Overexpression of the protein enhances the production of itaconic acid in microorganisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the national phase of PCT applicationPCT/NL2009/050099 having an international filing date of 4 Mar. 2009,which claims benefit of European patent application No. 08152332.6 filed5 Mar. 2008. The contents of the above patent applications areincorporated by reference herein in their entirety.

Reference to Sequence Listing Submitted Via EFS-WEB

The entire content of the following electronic submission of thesequence listing via the USPTO EFS-WEB server, as authorized and setforth in MPEP §1730 II.B.2(a)(C), is incorporated herein by reference inits entirety for all purposes. The sequence listing is identified on theelectronically filed text file as follows:

File Name Date of Creation Size (bytes) 313632010000Seqlist.txt Nov. 29,2010 50,602 bytes

The invention relates to the field of microbial production, morespecifically production of itaconic acid (itaconate), more specificallyproduction of itaconate in micro-organisms.

Production and metabolism of itaconic acid in microbial cells has beenstudied extensively for several decades (Calam, C. T. et al., 1939,Thom. J. Biochem., 33:1488-1495; Bentley, R. and Thiessen, C. P., 1956,J. Biol. Chem. 226:673-720; Cooper, R. A. and Kornberg, H. L., 1964,Biochem. J., 91:82-91; Bonnarme, P. et al., 1995, J. Bacteriol.117:3573-3578; Dwiarti, L. et al., 2002, J. Biosci. Bioeng. 1:29-33),but the metabolic pathway for itaconic acid has not been unequivocallyestablished (Wilke, Th. and Vorlop, K.-D., 2001, Appl. Microbiol.Biotechnol. 56:289-295; Bonnarme, P. et al., 1995, J. Bacteriol.177:3573-3578). Two complicating factors in this respect are that thebiosynthesis route for itaconic acid is thought to occur both in thecytosol and the mitochondria (Jaklitsch, W. M. et al., 1991, J. Gen.Microbiol. Appl. 6:51-61) and that aconitase, the enzyme thatinterconverts citric acid into cis-aconitate, and vice versa, and otherenzymes in the metabolic pathway have been found to be present in manyisoforms in microbial cells.

Production of itaconic acid is now commercially achieved in Aspergillusterreus, which has physiological similarity to A. niger and A. oryzae.However, these latter two accumulate citric acid, due to the absence ofcis-aconic acid decarboxylase (CAD) activity. Substrates used by thesefungi include mono- and disaccharides, such as glucose, sucrose andfructose and starches, as they exist in forms that are degradable by themicro-organism, and molasses. Recently, it has been discovered that alsoglycerol is a useful substrate in itaconic acid production by A. terreus(U.S. Pat. No. 5,637,485).

The general scheme currently envisioned for itaconic acid biosynthesisis given in FIG. 1, wherein clearly the existence of the biosyntheticroute both in the cytosol and the mitochondria is depicted and theconnection between these two compartments. At several points of thisscheme possibilities exist to try to improve the existing commercialproduction of itaconic acid in micro-organisms.

SUMMARY OF THE INVENTION

The invention comprises a nucleic acid sequence encoding an itaconatetransporting Major Facilitator Superfamily Transporter (MFST) genesequence (hereinafter “the itaconate transporter”). Preferably saidnucleic acid comprises the ATEG_(—)09972.1 sequence of Aspergillusterreus as depicted in FIG. 3B, or a nucleic acid that shares more thanabout 70%, preferably more than about 80%, preferably more than about90% sequence identity with the sequence of ATEG_(—)09972.1 as depictedin FIG. 3 b. In another embodiment, the invention comprises a proteinencoded by said nucleic acid.

The invention further comprises a method for the production of itaconicacid. More specifically the invention relates to improved production ofitaconic acid, comprising increasing the activity of a protein capableof transporting itaconate from the cytosol to the extracellular medium,in a suitable host cell. Preferably this is achieved by overexpressionof a nucleic acid sequence encoding the protein of the invention.Preferably the said nucleic acid is derived from Aspergillus sp. suchas, Aspergillus terreus, Aspergillus niger, Aspergillus nidulans,Aspergillus oryzae or Aspergillus fuminagates. According to a furtherpreferred embodiment, the said nucleic acid is expressed in a suitablevector, under control of its own or other promoters.

Also comprised in the invention is a method as described above, whereinthe above described transport of itaconic acid is further increased byincreasing the intracellular itaconic acid, using at least one butpreferably a combination of the following methods: 1. overexpression ofthe gene coding for the enzyme CAD (see EP 07112895) which catabolisescis-aconitate to itaconic acid, preferably wherein said gene is encodedby the nucleic acid sequence of ATEG 09971.1; 2. overexpression of agene coding for a protein capable of transporting di/tricarboxylate,preferably cis-aconitate, citrate or isocitrate, from the mitochondrionto the cytosol, more preferably the diacrboxylate transporter encoded bythe nucleic acid sequence of ATEG 09970.1 (see EP 08151584); 3. a methodas described above, wherein the activity of a regulator protein thatcomprises a zinc finger and a fungal specific transcription factordomain is modulated. Preferably said regulator protein is the proteinencoded by the nucleic acid sequence of ATEG 09969.1, located in thesame gene cluster as the transporter of the invention. By using theabove method 1 also organisms that do not or hardly produce itaconicacid like A. niger and A. oryzae due to the absence of endogenouscis-aconic acid decarboxylase (CAD) activity can be used sinceexpression of the CAD gene will cause itaconic acid production.

Another embodiment of the present invention is formed by a host cellwherein a gene coding for an itaconate transporter is introduced.Preferably said gene comprises the nucleotide sequence of the inventionencoding a transporter protein. A suitable host cell preferably is ahost cell selected from filamentous fungi, yeasts and bacteria, morepreferably from Escherichia coli, Aspergillus sp such as Aspergillusniger or Aspergillus terreus, citrate-producing hosts or lovastatinproducing hosts. The invention further comprises a host cell asdescribed above wherein the gene coding for a protein capable oftransporting di/tricarboxylate, preferably cis-aconitate, citrate orisocitrate, from the mitochondrion to the cytosol, is co-expressed. Theinvention further comprises a host cell as described above, wherein thetransported or produced cis-aconitate is catabolised to itaconic acid byoverexpression of the gene encoding the enzyme CAD.

Further, the invention pertains to the use of the protein(s)transporting itaconate from the cytosol to the extracellular medium, forthe production of itaconic acid by a suitable host cell. Also comprisedin the invention is the use of said protein(s) combined with theproteins transporting di/tricarboxylate over the mitochondrial membrane,the regulator protein ATEG 09969.2 and/or the use of the CAD enzyme, forthe production of itaconic acid in a suitable host cell.

LEGENDS TO THE FIGURES

FIG. 1: Postulated biosynthesis route(s) for itaconic acid in A.terreus. 1, Citrate synthase; 2, Aconitase; 3, cis-aconitic aciddecarboxylase (itaconate-forming); 4, cis-aconitic acid decarboxylase(citraconate-forming); 5, citraconate isomerase; 6, mitochondrialdicarboxylate-tricarboxylate antiporter; 7, mitochondrial tricarboxylatetransporter; 8, dicarboxylate transporter; 9, 2-methylcitratedehydratase.

FIG. 2: Overview of Aspergillus terreus genome segment with the clusterof genes involved in production of itaconic acid and lovastatin rangingfrom ATEG 09961.1-ATEG 09975.1. The cluster contains the cis-aconitatedecarboxylase (ATEG 09971.1) and the mitochondrial tricarboxylatetransporter (ATEG 9970.1).

FIG. 3: Sequence of the Aspergillus terreus itaconate transporter: a.protein sequence (SEQ ID NO:3), b. Cdna (SEQ ID NO:2), c. genomicsequence (SEQ ID NO:1).

FIG. 4: Sequence of the Aspergillus oryzae itaconate transporter: a.protein sequence (SEQ ID NO:6), b. cDNA (SEQ ID NO:5), c. genomicsequence (SEQ ID NO:4).

DETAILED DESCRIPTION OF THE INVENTION

“Fungi” are herein defined as eukaryotic micro-organisms and include allspecies of the subdivision Eumycotina (Alexopoulos, C. J., 1962, In:Introductory Mycology, John Wiley & Sons, Inc., New York). The termfungus thus includes both filamentous fungi and yeast. “Filamentousfungi” are herein defined as eukaryotic micro-organisms that include allfilamentous forms of the subdivision Eumycotina. These fungi arecharacterized by a vegetative mycelium composed of chitin, cellulose,and other complex polysaccharides. The filamentous fungi used in thepresent invention are morphologically, physiologically, and geneticallydistinct from yeasts. Vegetative growth by filamentous fungi is byhyphal elongation and carbon catabolism of most filamentous fungi areobligately aerobic. “Yeasts” are herein defined as eukaryoticmicro-organisms and include all species of the subdivision Eumycotinathat predominantly grow in unicellular form. Yeasts may either grow bybudding of a unicellular thallus or may grow by fission of the organism.

The term “fungal”, when referring to a protein or nucleic acid moleculethus means a protein or nucleic acid whose amino acid or nucleotidesequence, respectively, naturally occurs in a fungus.

The term “gene”, as used herein, refers to a nucleic acid sequencecontaining a template for a nucleic acid polymerase, in eukaryotes, RNApolymerase II. Genes are transcribed into mRNAs that are then translatedinto protein.

“Expression” refers to the transcription of a gene into structural RNA(rRNA, tRNA) or messenger RNA (mRNA) with subsequent translation into aprotein.

The term “vector” as used herein, includes reference to an autosomalexpression vector and to an integration vector used for integration intothe chromosome.

The term “expression vector” refers to a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestunder the control of (i.e., operably linked to) additional nucleic acidsegments that provide for its transcription. Such additional segmentsmay include promoter and terminator sequences, and may optionallyinclude one or more origins of replication, one or more selectablemarkers, an enhancer, a polyadenylation signal, and the like. Expressionvectors are generally derived from plasmid or viral DNA, or may containelements of both. In particular an expression vector comprises anucleotide sequence that comprises in the 5′ to 3′ direction andoperably linked: (a) a yeast-recognized transcription and translationinitiation region, (b) a coding sequence for a polypeptide of interest,and (c) a yeast-recognized transcription and translation terminationregion. “Plasmid” refers to autonomously replicating extrachromosomalDNA which is not integrated into a microorganism's genome and is usuallycircular in nature.

An “integration vector” refers to a DNA molecule, linear or circular,that can be incorporated in a microorganism's genome and provides forstable inheritance of a gene encoding a polypeptide of interest. Theintegration vector generally comprises one or more segments comprising agene sequence encoding a polypeptide of interest under the control of(i.e., operably linked to) additional nucleic acid segments that providefor its transcription. Such additional segments may include promoter andterminator sequences, and one or more segments that drive theincorporation of the gene of interest into the genome of the targetcell, usually by the process of homologous recombination. Typically, theintegration vector will be one which can be transferred into the hostcell, but which has a replicon that is nonfunctional in that organism.Integration of the segment comprising the gene of interest may beselected if an appropriate marker is included within that segment.

“Transformation” and “transforming”, as used herein, refer to theinsertion of an exogenous polynucleotide into a host cell, irrespectiveof the method used for the insertion, for example, direct uptake,transduction, f-mating or electroporation. The exogenous polynucleotidemay be maintained as a non-integrated vector, for example, a plasmid, oralternatively, may be integrated into the host cell genome.

By “host cell” is meant a cell that contains a vector or recombinantnucleic acid molecule and supports the replication and/or expression ofthe vector or recombinant nucleic acid molecule. Host cells may beprokaryotic cells such as E. coli, or eukaryotic cells such as yeast,fungus, plant, insect, amphibian, or mammalian cells. Preferably, hostcells are fungal cells.

Key in the biosynthetic pathway for itaconic acid is the localisation ofthe various substrates. It is thought that production of itaconic acidmainly occurs in the cytosol (see FIG. 1). In many biochemical pathways,the end-product is inhibiting its own production to prevent excessend-product in the biological system. Excess end-product will not onlylead to loss of energy in an economical sense, it can also give rise tounwanted side effects such as toxicity. It is contemplated that bydepleting the cell of itaconic acid the formation of new itaconic acidwill continue without end-product inhibition, thus giving—in total—anincrease yield of itaconic acid. Additionally the present inventionenables a more simple way of harvesting the itaconic acid due to itspresence in the extracellular medium. This also enables continuousfermentation culture.

Also provided are functional homologues of the ATEG_(—)09972.1sequences, that are 50% or more identical to the sequence of FIG. 3 b,preferably 60% or more, more preferably 70% or more, more preferably 80%or more, more preferably 90% or more and most preferably 95% or moreidentical. Functional in the term functional homologues means that thehomologous protein has an itaconic acid/itaconate transporter functioni.e. is able to transport itaconate over the cell membrane.

The term “sequence identity,” as used herein, is generally expressed asa percentage and refers to the percent of amino acid residues ornucleotides, as appropriate, that are identical as between two sequenceswhen optimally aligned. For the purposes of this invention, sequenceidentity means the sequence identity determined using the well-knownBasic Local Alignment Search Tool (BLAST), which is publicly availablethrough the National Cancer Institute/National Institutes of Health(Bethesda, Md.) and has been described in printed publications (see,e.g., Altschul et al., J. MoI. Biol, 215(3), 403-10 (1990)). Preferredparameters for amino acid sequences comparison using BLASTP are gap open11.0, gap extend 1, Blosum 62 matrix.

Every nucleic acid sequence herein that encodes a polypeptide also, byreference to the genetic code, describes every possible silent variationof the nucleic acid. The term “conservatively modified variants” appliesto both amino acid and nucleic acid sequences. With respect toparticular nucleic acid sequences, conservatively modified variantsrefers to those nucleic acids which encode identical or conservativelymodified variants of the amino acid sequences due to the degeneracy ofthe genetic code.

The term “degeneracy of the genetic code” refers to the fact that alarge number of functionally identical nucleic acids encode any givenprotein. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations” and represent onespecies of conservatively modified variation.

The present inventors have found a method for increasing the productionof itaconic acid, by increasing the activity of the protein capable oftransporting itaconate from the cytosol to the extracellular medium,leading to increased recovery of itaconic acid, produced by a suitablemicro-organism. The protein is further defined as a protein capable oftransporting itaconic acid.

Preferably, the itaconate transporter is the itaconate transporter ofAspergillus terreus, as is found in the itaconate/lovastatin genecluster (see FIG. 2), which is represented by the nucleic acid sequencefound in ATEG_(—)09972.1, which is disclosed in FIGS. 3 b and 3 c, forthe cDNA and genomic sequence, respectively.

Further example of an itaconate transporter that could also be used inthe present invention is the homologous gene denominated BAE57135.1 fromA. oryzae, as depicted in FIGS. 4 a, 4 b and 4 c. The produced itaconicacid can be recovered from the extracelluar medium using methods know toa person skilled in the art and as described by Wilke et al. (Wilke, Th.and Vorlop, K.-D., 2001, Appl. Microbiol. Biotechnol. 56:289-295). Onepreferred way of increasing the activity of said protein(s) is byoverexpression of a gene(s) encoding said protein(s), preferably whereinsaid gene is ATEG_(—)09972.1.

Overexpression can be effected in several ways. It can be caused bytransforming the micro-organism with a gene coding for the enzyme.Alternatively, other methods can be used for effecting an increase inthe activity of said enzyme. One possible way is to provide a strongerpromoter in front of and regulating expression of the endogenous gene.This can be achieved by use of a strong heterologous promoter or byproviding mutations in the endogenous promoter. An increased activity ofthe enzyme can also be caused by removing possible inhibiting regulatoryproteins, e.g. by inhibiting the expression of such proteins. The personskilled in the art will know other ways of increasing the activity ofthe above mentioned enzyme.

The production of itaconic acid can be further optimised by combiningthe product secretion and recovery as described above, withoverexpression of di/tricarboxylate transporters, capable oftransporting, among others, cis-aconitate, citrate or isocitrate fromthe mitochondrion to the cytosol, preferably the gene encoded by thenucleic acid sequence of ATEG_(—)09970.1. These subsequent processeswill lead to an increase in cis-aconitate in the cytosol, which can befurther converted to itaconic acid, using overexpression of the geneencoding the enzyme CAD (EC 4.1.1.6). “CAD” is defined as the enzyme, ora nucleotide sequence encoding for the enzyme cis-aconitatedecarboxylase (CAD), such as the enzyme encoded by the nucleic acidsequence of ATEG_(—)09971.1, this further comprises enzymes with similaractivities (see EP07112895).

Even further optimisation of the present invention can be achieved bymodulating the activity of the regulator protein that comprises a zincfinger and a fungal specific transcription factor domain as can be foundon the gene cluster that also comprises ATEG_(—)09970, wherein thisregulator protein is indicated as ATEG_(—)09969.1 (see FIG. 2).

The above described processes alone or in combination lead to asubsequent increase of itaconic acid (see FIG. 1). Another advantage ofthe invention is an improved method to recover the produced itaconicacid by increasing the release in the extracellular media. Thecombination of improved production and improved recovery leads to anincrease in itaconic acid yield by a suitable host. The above describedgenes are preferably derived from Aspergillus sp. like, Aspergillusterreus, Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae orAspergillus fuminagates. However, it is also possible to derive the genefrom other itaconate producing micro-organisms such as Ustilago zeae,Ustilago maydis, Ustilago sp., Pseudozyma antarctica, Candida sp.,Yarrowia lipolytica, and Rhodotorula sp.

In another aspect of the invention, micro-organisms overexpressing atleast one but alternatively a combination of the above mentionednucleotide sequences, encoding at least a protein capable oftransporting itaconate from the cytosol to the extracellular medium, areproduced and used for increased production of itaconic acid. Morepreferably micro-organisms overexpressing a protein that transportsitaconate combined with protein(s) that transport di/tricarboxylatesfrom the mitochondrion to the cytosol and/or the CAD enzyme are used tofurther improve the production of itaconic acid.

Micro-organisms used in the invention are preferably micro-organismsthat naturally produce itaconic acid. Preferably overexpression of thegenes encoding the above described protein(s) and enzyme(s) isaccomplished in filamentous fungi, yeasts and/or bacteria, such as, butnot limited to Aspergillus sp., such as the fungi A. terreus, A.itaconicus and A. niger, Aspergillus nidulans, Aspergillus oryzae orAspergillus fuminagates, Ustilago zeae, Ustilago maydis, Ustilago sp.,Candida sp., Yarrowia lipolytica, Rhodotorula sp. and Pseudozymaantarctica, the bacterium E. coli and the yeast Saccharomycescerevisiae. Especially preferred are heterologous citric acid producingorganisms in which the substrates are available in the host organism.

Recently (see US 2004/0033570) it has also been established that theso-called D4B segment of Aspergillus terreus, which comprises the CADgene is responsible for the synthesis of lovastatin (see FIG. 2). Thus,it is submitted that also these micro-organisms which are known toproduce lovastatin would be suitable candidates for the production ofitaconic acid. Such micro-organisms include Monascus spp. (such as M.ruber, M. purpureus, M. pilosus, M. vitreus and M. pubigerus),Penicillium spp. (such as P. citrinum, P. chrysogenum), Hypomyces spp.,Doratomyces spp. (such as D. stemonitis), Phoma spp., Eupenicilliumspp., Gymnoascus spp., Pichia labacensis, Candida cariosilognicola,Paecilomyces virioti, Scopulariopsis brevicaulis and Trichoderma spp.(such as T. viride).

Consequently also the CAD encoding part of the D4B segment and theenzyme with CAD activity for which it codes from these above-mentionedlovastatin producing micro-organisms are deemed to be suitable for usein the present invention. It further is contemplated that a heterologousorganism, which in nature does not or hardly produce itaconic acid likeAspergillus niger or Aspergillus oryzae can be used when providing suchan organism with a functional pathway for expression of itaconic acid,by overexpression of the above mentioned genes.

Recombinant host cells described above can be obtained using methodsknown in the art for providing cells with recombinant nucleic acids.These include transformation, transconjugation, transfection orelectroporation of a host cell with a suitable plasmid (also referred toas vector) comprising the nucleic acid construct of interestoperationally coupled to a promoter sequence to drive expression. Hostcells of the invention are preferably transformed with a nucleic acidconstruct as further defined below and may comprise a single butpreferably comprises multiple copies of the nucleic acid construct. Thenucleic acid construct may be maintained episomally and thus comprise asequence for autonomous replication, such as an ARS sequence. Suitableepisomal nucleic acid constructs may e.g. be based on the yeast 2μ orpKD1 (Fleer et al., 1991, Biotechnology 9: 968-975) plasmids.Preferably, however, the nucleic acid construct is integrated in one ormore copies into the genome of the host cell. Integration into the hostcell's genome may occur at random by illegitimate recombination butpreferably the nucleic acid construct is integrated into the host cell'sgenome by homologous recombination as is well known in the art of fungalmolecular genetics (see e.g. WO 90/14423, EP-A-0 481 008, EP-A-0 635 574and U.S. Pat. No. 6,265,186) Most preferably for homologousrecombination the ku70Δ/ku80Δ techniques is used as described forinstance in WO 02/052026 and Krappmann, 2007, Fungal Biol. Rev.21:25-29).

Transformation of host cells with the nucleic acid constructs of theinvention and additional genetic modification of the fungal host cellsof the invention as described above may be carried out by methods wellknown in the art. Such methods are e.g. known from standard handbooks,such as Sambrook and Russel (2001) “Molecular Cloning: A LaboratoryManual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, or F. Ausubel et al, eds., “Current protocols inmolecular biology”, Green Publishing and Wiley Interscience, New York(1987). Methods for transformation and genetic modification of fungalhost cells are known from e.g. EP-A-0 635 574, WO 98/46772, WO 99/60102and WO 00/37671.

In another aspect the invention relates to a nucleic acid constructcomprising a nucleotide sequence encoding at least an itaconatetransporter as defined above and usable for transformation of a hostcell as defined above. In the nucleic acid construct, the codingnucleotide sequence(s) preferably is/are operably linked to a promoterfor control and initiation of transcription of the nucleotide sequencein a host cell as defined below. The promoter preferably is capable ofcausing sufficient expression of the itaconate transporters transportingitaconate from the cytosol to the extracellular medium and/or thedi/tricarboxylate transporters transporting tricarboxylates from themitochondrion and/or the CAD enzyme(s), described above, in the hostcell. Promoters useful in the nucleic acid constructs of the inventioninclude the promoter that in nature provides for expression of thecoding genes. Further, both constitutive and inducible natural promotersas well as engineered promoters can be used. Promoters suitable to driveexpression of the genes in the hosts of the invention include e.g.promoters from glycolytic genes (e.g. from a glyceraldehyde-3-phosphatedehydrogenase gene), ribosomal protein encoding gene promoters, alcoholdehydrogenase promoters (ADH1, ADH4, and the like), promoters from genesencoding amylo- or cellulolytic enzymes (glucoamylase, TAKA-amylase andcellobiohydrolase). Other promoters, both constitutive and inducible andenhancers or upstream activating sequences will be known to those ofskill in the art. The promoters used in the nucleic acid constructs ofthe present invention may be modified, if desired, to affect theircontrol characteristics. Preferably, the promoter used in the nucleicacid construct for expression of the genes is homologous to the hostcell in which genes are expressed.

In the nucleic acid construct, the 3′-end of the coding nucleotide acidsequence(s) preferably is/are operably linked to a transcriptionterminator sequence. Preferably the terminator sequence is operable in ahost cell of choice. In any case the choice of the terminator is notcritical; it may e.g. be from any fungal gene, although terminators maysometimes work if from a non-fungal, eukaryotic, gene. The transcriptiontermination sequence further preferably comprises a polyadenylationsignal.

Optionally, a selectable marker may be present in the nucleic acidconstruct. As used herein, the term “marker” refers to a gene encoding atrait or a phenotype which permits the selection of, or the screeningfor, a host cell containing the marker. A variety of selectable markergenes are available for use in the transformation of fungi. Suitablemarkers include auxotrophic marker genes involved in amino acid ornucleotide metabolism, such as e.g. genes encodingornithine-transcarbamylases (argB), orotidine-5′-decarboxylases (pyrG,URA3) or glutamine-amido-transferase indoleglycerol-phosphate-synthasephosphoribosyl-anthranilate isomerases (trpC), or involved in carbon ornitrogen metabolism, such e.g. niaD or facA, and antibiotic resistancemarkers such as genes providing resistance against phleomycin, bleomycinor neomycin (G418). Preferably, bidirectional selection markers are usedfor which both a positive and a negative genetic selection is possible.Examples of such bidirectional markers are the pyrG (URA3), facA andamdS genes. Due to their bidirectionality these markers can be deletedfrom transformed filamentous fungus while leaving the introducedrecombinant DNA molecule in place, in order to obtain fungi that do notcontain selectable markers. This essence of this MARKER GENE FREE™transformation technology is disclosed in EP-A-0 635 574, which isherein incorporated by reference. Of these selectable markers the use ofdominant and bidirectional selectable markers such as acetamidase geneslike the amdS genes of A. nidulans, A. niger and P. chrysogenum is mostpreferred. In addition to their bidirectionality these markers providethe advantage that they are dominant selectable markers that, the use ofwhich does not require mutant (auxotrophic) strains, but which can beused directly in wild type strains.

Optional further elements that may be present in the nucleic acidconstructs of the invention include, but are not limited to, one or moreleader sequences, enhancers, integration factors, and/or reporter genes,intron sequences, centromers, telomers and/or matrix attachment (MAR)sequences. The nucleic acid constructs of the invention may furthercomprise a sequence for autonomous replication, such as an ARS sequence.Suitable episomal nucleic acid constructs may e.g. be based on the yeast2μ or pKD1 (Fleer et al., 1991, Biotechnology 9: 968-975) plasmids.Alternatively the nucleic acid construct may comprise sequences forintegration, preferably by homologous recombination (see e.g.WO98/46772). Such sequences may thus be sequences homologous to thetarget site for integration in the host cell's genome. The nucleic acidconstructs of the invention can be provided in a manner known per se,which generally involves techniques such as restricting and linkingnucleic acids/nucleic acid sequences, for which reference is made to thestandard handbooks, such as Sambrook and Russel (2001) “MolecularCloning: A Laboratory Manual (3rd edition), Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al,eds., “Current protocols in molecular biology”, Green Publishing andWiley Interscience, New York (1987).

In a further aspect the invention relates to fermentation processes inwhich the transformed host cells of the invention are used for theconversion of a substrate into itaconic acid. A preferred fermentationprocess is an aerobic fermentation process. The fermentation process mayeither be a submerged or a solid state fermentation process.

In a solid state fermentation process (sometimes referred to assemi-solid state fermentation) the transformed host cells are fermentingon a solid medium that provides anchorage points for the fungus in theabsence of any freely flowing substance. The amount of water in thesolid medium can be any amount of water. For example, the solid mediumcould be almost dry, or it could be slushy. A person skilled in the artknows that the terms “solid state fermentation” and “semi-solid statefermentation” are interchangeable. A wide variety of solid statefermentation devices have previously been described (for review see,Larroche et al., “Special Transformation Processes Using Fungal Sporesand Immobilized Cells”, Adv. Biochem. Eng. Biotech., (1997), Vol 55, pp.179; Roussos et al., “Zymotis: A large Scale Solid State Fermenter”,Applied Biochemistry and Biotechnology, (1993), Vol. 42, pp. 37-52;Smits et al., “Solid-State Fermentation-A Mini Review, 1998),Agro-Food-Industry Hi-Tech, March/April, pp. 29-36). These devices fallwithin two categories, those categories being static systems andagitated systems. In static systems, the solid media is stationarythroughout the fermentation process. Examples of static systems used forsolid state fermentation include flasks, petri dishes, trays, fixed bedcolumns, and ovens. Agitated systems provide a means for mixing thesolid media during the fermentation process. One example of an agitatedsystem is a rotating drum (Larroche et al., supra). In a submergedfermentation process on the other hand, the transformed fungal hostcells are fermenting while being submerged in a liquid medium, usuallyin a stirred tank fermenter as are well known in the art, although alsoother types of fermenters such as e.g. airlift-type fermenters may alsobe applied (see e.g. U.S. Pat. No. 6,746,862).

Preferred in the invention is a submerged fermentation process, which isperformed in a fed-batch or repeated (fed-) batch mode. In a fed-batchfermentation there is a continuous input of feed containing a carbonsource and/or other relevant nutrients in order to improve itaconic acidyields. The input of the feed can, for example, be at a constant rate orwhen the concentration of a specific substrate or fermentation parameterfalls below some set point. In a repeated batch fermentation the cultureis harvested at regular time-intervals by stopping the fermentation andretrieving the produced product from the medium. Next to refreshing themedium often also part of the microbial culture is discarded, while therest is used as a new inoculum for a following batch culture.

It is preferred to use a host cell that naturally would contain theenzymes/transporters of the itaconic acid pathway as depicted in FIG. 1,and the enzymes/transporters of the citric acid pathways in the cytosoland mitochondrion. However, if the host would lack one or more of thesegenes, they can be co-introduced with the above described enzymes andproteins. Such a co-introduction can be performed by placing thenucleotide sequence of such a gene on the same plasmid vector as theabove described genes, or on a separate plasmid vector.

Further, since the itaconic acid pathway is located partly in thecytosol and partly in the mitochondrion, it is contemplated thatoverexpression of the genes/enzymes in either or both of thosecompartments would be desirable. The person skilled in the art will knowhow to achieve overexpression in the cytosol or mitochondria by usingthe appropriate signal sequences.

EXAMPLES Example 1 Construction of Micro-Array

An anonymous clone/EST-based array approach was taken according to thefollowing scheme:

An A. terreus micro-array was made composed of a clone-based and anEST-based array.Materials and Methods Construction Micro-ArrayIsolation of Chromosomal DNA from A. Terreus

A. terreus was cultivated overnight in a shake flask in enriched minimalmedium at 33° C. and 250 rpm. Enriched minimal medium (pH 5.5) ismineral medium (MM) supplemented with 0.5% yeast extract and 0.2%casamino acids. The composition of MM was: 0.07 M NaNO₃, 7 mM KCl, 0.11M KH₂PO₄, 2 mM MgSO₄, and 1 ml/l of trace elements (1000*stock solution:67 mM ZnSO₄, 178 mM H₃BO₃, 25 mM MnCl₂, 18 mM FeSO₄, 7.1 mM CoCl₂, 6.4mM CuSO₄, 6.2 mM Na₂MoO₄, 174 mM EDTA).

Mycelium was harvested after 22 hours and frozen in liquid nitrogen.Chromosomal DNA was isolated from 4.5 g mycelium following the protocoldescribed below.

-   -   Grind 0.5-1.0 g mycelium under liquid nitrogen using the        membrane disrupter.        -   Place polypropylene tubes (Greiner) with 1.5 ml            water-saturated phenol, 1 ml TNS, 1 ml PAS and 0.5 ml 5×RNB            in a water bath at 55° C., add the still frozen mycelium to            the tubes and vortex every 20 seconds for totally 2-4            minutes.        -   TNS: triisopropyl naphthalene sulphonic acid, 20 mg/ml in            water, freshly prepared        -   PAS: 4 aminosalisylic acid, 120 mg/ml in water, freshly            prepared        -   5×RNB: 60.55 g Tris, 36.52 g NaCl, 47.55 g EGTA in 500 ml            water (pH=8.5)    -   Add 1 ml sevag (24:1 chloroform:isoamyl alcohol) and vortex with        intervals for another 1-2 minutes.    -   Spin for 10 min. in the tabletop centrifuge at 4° C. at maximum        velocity.    -   Extract the water-phase once again with phenol-sevag and twice        with sevag. GENTLY, AVOID SHEARING!    -   Precipitate the DNA with 2 volumes ethanol. Spin directly for 10        min. in the tabletop centrifuge.    -   Drain the tube, dry it with Kleenex and resuspend the pellet in        500 μl Tris/EDTA. Transfer to a microvial.    -   Extract with phenol-sevag until interface stays clean. Then        extract once with sevag.    -   Precipitate with 2 volumes ice-cold ethanol, spin down and        resuspend the pellet in 100-200 μl TE with 50 μg/ml RNase.        Construction of Clone-Based gDNA Library        The gDNA library was prepared as follows:

Chromosomal A. terreus DNA was sheared into fragments of size 1.5-2.5 kb

-   -   The sheared DNA was subsequently size fractionated, end-repaired        (Lucigen), and ligated into blunt-end pSMART-HC-Amp vectors        (Lucigen).    -   The ligated constructs were transformed into E. coli DH 10b    -   Colony PCR was performed on 96 transformants to check that >90%        of the inserts had the correct size    -   Sequence analysis (short run) was performed on 20 clones to        confirm their diversity and fungal origin    -   Colony picking of 20,000 amp-resistant colonies was carried out        into 96-well microtiter plates containing TY medium+100 μg/ml        ampicillin        The 20.000 clones were replicated into 96-well microtiter        plates. The ordered libraries are stored as glycerol stocks at        −80° C.        Generation of mRNA for cDNA Library Construction        Precultures: A. terreus spores (10⁶-10⁷/ml) were inoculated into        100 ml B medium (2 g/l NH4NO3; 1 g/l MgSo4*7H2O; 0.008 g/l        ZnSO4*7H2O; 0.015 g/l CuSO4*5H2O; 1.5 ppm FeSO4*5H2O; 0.08 g/l        KH2PO4; 10 g/l CaCl2*2H2O, set to pH 3.1 with HCl) containing 20        g/l glucose, and incubated for 24-48 hours at 37° C. at 250 rpm.        Production cultures (B medium containing 100 g/l glucose) were        inoculated 1/10 (v/v) for 2-days cultivations and 1/25 (v/v) for        3-day cultivations. After 2-3 days cultivation mycelium was        harvested, filtered over miracloth, washed with 0.2 M sodium        phosphate buffer (pH 6.5), frozen in liquid nitrogen and stored        at −80° C.        Isolation of mRNA from A. Terreus    -   grind mycelium with mortar and pestle under liquid nitrogen; add        100 μl β-mercaptoethanol before grinding to inactivate RNAse    -   transfer powder to cooled plastic tube (1.0 g per tube); keep        mycelium frozen    -   add 4 ml Trizol and vortex till homogenous    -   add 0.4 ml chloroform and vortex    -   centrifuge for 20-30 min. at 3700 rpm, 4° C.    -   transfer supernatant to Eppendorf tubes (1.2 ml per tube)    -   add 0.7 ml per 1.2 ml supernatant    -   centrifuge in eppendorf centrifuge for 15 min. at 14.000 rpm, 4°        C.    -   wash pellet with 1 ml 70% ethanol    -   centrifuge 5 min., 14.000 rpm, 4° C.    -   air-dry pellet and resuspend in 0.2 ml water    -   store RNA samples at −80° C.        Construction of cDNA library        The cDNA library was prepared as follows:    -   The RNA was run on gel to determine the quality of the sample    -   polyT-primed cDNA was prepared from the total RNA provided        (RT-PCR reaction using superscript and dT primers    -   The cDNA was size fractionated to give fragments of size 1.0-1.5        kb    -   The fragments were end-repaired (Lucigen), and ligated into        blunt-end p SMART-HC-kan vectors (Lucigen).    -   Restriction analysis of 96 clones was performed to check the        insert size and the % of transformants which had the correct        insert size    -   Sequence analysis (short run) of 20 clones was performed to        confirm diversity and fungal origin    -   5,000 kanamycin-resistant colonies were picked into microtiter        plates        The 5000 cDNA clones were replicated into 96-well microtiter        plates. The ordered libraries were stored as glycerol stocks at        −80° C.        Construction of the A. Terreus Clone-Based Array

PCR fragments were generated from the different clones from the gDNA(20,000 clones) and cDNA (5,000 clones) library by mass 96 well PCR (50μl/well, Lucigen SMART-SR1/SL1 primers with 5′-C6-aminolinkers, SuperTaqand buffer from HT Biotech. Ltd, dNTP's (Roche 11 969 064 001), pintooldipped template from grown colony plates).

All above PCR products were purified by 96 well precipitation(isopropanol and 96% ethanol wash), speedvac dried, dissolved in 15 μl3×SSC/well and spotted with quill pins (Telechem SMP3) on CSS100silylated aldehyde glass slides (Telechem, USA) using a SDDC2Eurogridder (ESI, Canada). During spotting, aminolinkers of PCR productswill covalently link with aldehyde groups of the coated slides.

gDNA and cDNA PCR products were spotted on two separate slides (slide a:1st 10,000 gDNA's+5000 cDNA's; slide b: 2nd 10,000 gDNA's+same 5000cDNA's).

For the clone-based array a genomic library was constructed. A total of20,000 clones containing chromosomal fragments was generated, 90% ofwhich had an average insert size of 1.5-2.5 kb. This resulted in a fullgenome coverage of 64% (Akopyants et al., 2001).

For the EST-based array a cDNA library of in total 5000 cDNA clones wasconstructed, 70% of which had an average insert size of 1.0-1.5 kb. Thisso-called EST-based approach has the advantage that it will be enrichedfor the genes expressed under the selected (itaconic acid producing)conditions. Moreover, in the EST-based approach per clone (and thusspot) only a single gene is represented in eukaryotes.

The complete micro-array, thus consisting of 20,000 genomic DNA clonesand 5,000 cDNA clones was composed of an A and a B glass slide. Bothslides contained the same 5,000 cDNA spots. The A and B slide eachcontained 10,000 of the gDNA spots.

Example 2 Generation of the Different RNA Samples by Fermentation

Materials and Methods Fermentation and mRNA Isolation

Fermentation Conditions of A. Terreus

5-Liter controlled batch fermentations were performed in a New BrunswickScientific Bioflow 3000 fermentors. The following conditions were usedunless stated otherwise:

-   -   37° C.    -   pH start 3.5 set point 2.3    -   DO set points Day 1: 75%        -   Day 2, 3, 4: 50%        -   Subsequent days: 25%    -   Preculture: 100 ml of the same medium as used in the        fermentation medium (10⁷ spores/ml) in 500 ml Erlenmeyer flask        with baffles, overnight, 37° C., 150 rpm    -   pH control: 4M KOH (Base), 1.5 M H₃ PO₄ (Acid)    -   Antifoam: Struktol (Schill & Seilacher)        Fermentation Medium Compositions:        Per liter: 2.36 g of NH₄SO₄, 0.11 g of KH₂PO₄, 2.08 g of        MgSO₄*7H₂O, 0.13 g of CaCl₂*2H₂O, 0.074 g of NaCl, 0.2 mg of        CuSO₄*5H₂O, 5.5 mg of Fe(III) SO₄*7H₂O, 0.7 mg of MnCl₂*4H₂O and        1.3 mg of ZnSO₄*7H₂O and 100 g of glucose as a carbon source.        All media were prepared in demineralised water.        Isolation of mRNA from A. Terreus

See mRNA isolation protocol described in Example 1

Determination of the Itaconate Concentration by HPLC

5 μl of a 10-times diluted supernatant sample (split ratio 1:3) wasseparated using a Waters 2695 Separations module on a reversed-phaseDevelosil 3 μm RP-Aqueous C30 140A column (150×3 mm) (Phenomenex p/nCH0-6001) at 25° C. using the solvent gradient profile (flow rate was0.4 ml/min) shown in Table 1.

TABLE 1 Solvent gradient of the RP-UV method. A B Time (20 mM NaH₂PO₄(Acetonitril) (min) pH 2.25) (%) (%) 0 100 0 10 100 0 15 95 5 20 95 5 21100 0 30 100 0 Compounds were detected by UV at 210 nm using a Waters2487 Dual wavelength Absorbance detector (Milford, MA, USA).Itaconate Productivity

Itaconate productivity at a certain time point was calculated as theslope of the regression line between that particular time point and thetime points right before and after that time point. To this end of 6-10supernatant samples of the different fermentations, the itaconateconcentrations were determined by HPLC.

For the transcriptomics approach it is essential to have RNA samplesfrom fermentations that result in the production of different amounts ofitaconate. Therefore a literature survey was performed in order toidentify medium components and/or physicochemical conditions that affectthe amount of itaconate produced by A. terreus. Although manyconflicting reports were found regarding the effect that a specificparameter has on itaconic acid production, 4 key overall parameters wereidentified from this literature survey, i.e. (i) carbon source, (ii) pH,(iii) trace element (i.e. Mn) concentration and (iv) oxygen tension.Fermentations with A. terreus varying principally in these fourparameters were performed on a mineral salts medium to ensure that theelemental limitations required for itaconate production would beachieved. Table 2 presents an overview of the fermentations performed inthis study.

TABLE 2 Overview of the fermentations performed in order to generate RNAsamples for transcriptome analysis. Fermen- Max. Max. tation Fermen-Itaconic Biomass run tation Environmental condition acid (g/l) (gDWT/kg)First 1 Glucose (100 g/l) (control) 16.1 12.7 Run 2 Fructose as C-source8.84 13.7 3 Maltose as C-source 13.9 12.1 Second 4 Glucose (100 g/l) pHstart 25.8 11.6 run 3.5, set point 2.3 (control) 5 pH set 3.5 8.7 16.5 6pH start 3.5 no set point 30.6 8.7 Third 7 Low glucose (30 g/l) 11.1 6.7run 8 O₂ set point 25% 47.2 12.0 9 5* higher Mn 20.3 13.8 Fourth 10Glucose (100 g/l) (control) 26.9 17.9 run 11 pH set 4.5 0.1 20.4 12 O₂set point 10% 52.9 10.6 The reference fermentation is on 100 g/lglucose, dO2, day 1, 75%; day 2-4, 50%, day 5 and further 25%, pH start3.5, set point at 2.3.

As shown in Table 2, a considerable variation in the amount of itaconateis produced in this set of fermentations, ranging from almost noitaconate (fermentation #11; pH 4.5) to about 50 g/l itaconate (#8 and#12; 02 set point 25% and 10% respectively).

Of each fermentation 2 to 5 samples were harvested for isolation ofmRNA.

From in total 23 fermentation samples mRNA could be isolated. Of 7samples, mRNA was isolated twice independently. It proved to beespecially difficult (impossible) to extract RNA from the samples takenin the stationary phase. A number of samples showed partial degradationof the RNA. Although no mRNA could be isolated from the samples fromfermentations #6 and #12, the remaining samples still covered thecomplete range of itaconate production (Table 3).

TABLE 3 List of 30 mRNA samples from various fermentation conditionsthat were used for transcriptome analysis. The samples marked withasterix were the samples used for the differential expression dataanalysis. Itaconic Sample Fermentation RNA Itaconic acid acid RNA no.condition id EFT (hours) (g/l) Productivity quality R3 gluc100  1.3.a50.3 14.6 0.117 ok R4 gluc100  1.4.a 74.8 16.1 0.060 ok R5 fruc100 2.3.a 50.3  8.2 0.082 ok R6 fruc100  2.3.b 50.3  8.2 0.082 ok R7fruc100  2.4.a  75.05  8.6 −0.013 ok R8 malt100  3.3.a 50.3 7  0.355 okR9 malt100  3.4.a 75   12.1 0.220 ok R10 pH-i3.5  4.3.a  53.25 25.80.146 part degr R11 pH-i3.5  4.3.b  53.25 25.8 0.146 part degr R12pH-i3.5  4.4.a 73   24   −0.153* ok R13 pH-c3.5  5.3.a 53.5  7.5 −0.042ok R14 pH-c3.5  5.3.b 53.5  7.5 −0.042 ok R15 pH-c3.5  5.4.a  73.25  7.90.035 ok R16 gluc30  7.2.a  30.25 9  0.317 ok R1 gluc30  7.3.a 43.5 10  0.030 ok R17 gluc30  7.3.a 43.5 10   0.030 ok R18 O2s25%  8.2.a 30.536*  0.824* ok R19 O2s25%  8.4.a  78.25 46   0.029 part degr R20 5xMn 9.2.a  30.75 1  0.194 ok R21 5xMn  9.2.b  30.75 1  0.194 ok R22 5xMn 9.3.a 53.5 10   0.496 part degr R23 5xMn  9.3.b 53.5 10   0.496 partdegr R24 5xMn  9.4.a 78.5 19   0.189 part degr R25 5xMn  9.4.b 78.5 19  0.189 part degr R26 5xMn  9.5.a  93.25 20   0.106 ok R2 Gluc100 10.3.a51.5 14.7 0.256 ok R27 Gluc100 10.3.a 51.5 14.7 0.256 ok R28 Gluc10010.4.a 74   19.5 0.085 ok R29 Gluc100 10.5.a 100.4  22   0.177 part degrR30 Gluc100 10.5.b 100.4  22   0.177 part degr R31 pH 4.5 11.3.a 51.5  0.04* −0.001 ok R32 pH 4.5 11.4.a 74     0.05* 0.003 ok

Example 3 Transcriptome Analysis, Data Analysis of the Array Data

Materials and Methods Transcriptome Analysis, Data Normalization andData Analysis

Labeling of RNA and gDNA

Total RNA's (5 μg/30 μl reaction), isolated from various A. terreuscultures (strain NRRL 1960, BASF) with differential itaconateproduction, were labelled with amino-allyl-dUTP (0.75 μM aa-dUTP finalconc., Sigma A0410), using 3 μl 50 μM oligo p(dT)₁₅ primer (La Roche,814270), unlabelled dNTP's (added to 1.25 μM final conc. for each dNTP),2 μl Superscript II Reverse Transcriptase and buffer (Life Technologies,10297-018: primer annealing 10 min 70° C., transcriptase 180 min 42°).After RNA hydrolysis (3 μl 2.5M NaOH, 30 min 37°, 3 μl 2.5 M HAc) theaa-dUTP labelled cDNA was directly purified (below).

As a reference for correcting slide differences (spotting, labeling-,hybridization- and scan efficiency), gDNA (0.5 μg/reaction) ofAspergillus terreus (strain NRRL 1960, BASF) was labelled with aa-dUTP,using dNTP's (conc. as above), Klenov-DNA Polymerase and buffer(Bioprime kit, Invitrogen 18094-011: primer annealing 5 min 96° C.,polymerase 90 min 37°).

The aa-dUTP-labelled cDNA or gDNA was purified (QIAquick column, Qiagen28106), speedvac dried, dissolved (4.5 μl 0.1 M Na₂CO₃), coupled with4.5 μl Cy5-NHS-ester for cDNA, or 4.5 μl Cy3-NHS-ester for gDNA(Amersham/GE-Healthcare PA25001 or PA23001 respectively, each in 73 μlDMSO) for 60 min at 20° C., diluted with 10 μl of water, and againpurified on Autoseq G50 columns (GE-Healthcare 27-5340).

Array Blocking, (Pre) Hybridization and Image Analysis

Before hybridization with the array produced as described above, slideswere blocked (removal surplus of spotted PCR products and blocking offree aldehyde groups) by 3× quickly washing (20° C.) with Prehyb bufferand 45 min incubation (42° C.) in PreHyb buffer (5×SSC, 1% BSA, 0.1%SDS). After 4 washes in water, spotted PCR products were denatured bydipping the slides 5 sec in boiling water and drying them with aN₂-gas-pistol.

The Cy5- and Cy3-labelled sample were combined, 8 μl 25 μg/μl yeast tRNA(Invitrogen, 15401-029) and 4 μl 5 μg/μl poly-dA/dT (Amersham 27-7860)were added, the mixture was speed vac dried, dissolved in 160 μl Easyhybbuffer (Roche, 1 796 895), denatured (2 min, 96° C.), cooled to 50° C.,applied on a pair of prehybridised slides (a+b, 80 μl/slide) prewarmedat 50° C., covered with a cover slide (Hybri slibs, Mol. Probes.H-18201) and incubated overnight at 42° C. in a humidified hybridizationchamber (Corning 2551). Slides were washed (pair a+b in one 50 ml tube,1× in 1×SSC/0.1% SDS 37° C., 1× in 0.5×SSC 37° C., 2× in 0.2×SSC 20° C.)and dried with N₂-gas. All pre-hybridisation buffers were 0.45 μmfiltered to reduce dust noise. Slide images of Cy5- and Cy3-fluorescenceintensity (ScanArray Express Scanner & Software, Packard Biosc.) wereanalysed (Imagene 5.6 Software, Biodiscovery) to obtain for each spotsignal- and local background value (medians) for the hybridized Cy5-RNAand Cy3-reference gDNA. These values were used for further dataanalysis.

Array Data Normalization

Before normalization, all low abundant spots having a Signal/Backgroundbelow 1.5 were removed. Data were normalized using a total cDNA signalcorrection. For each slide and each spot, the difference between signaland background was calculated for Cy5 and Cy3. Per slide, the sum of thedifferences was taken for Cy5 and Cy3, and the ratio between these twowas used as normalisation factor for that particular slide. All spots(chromosomal and genomic) were normalised using this total cDNA signal.

Data Analysis of the Transcriptomics Data by Multivariate RegressionAnalysis

Scaling

Data were range scaled [(x− x)/(x_(max)−x_(min))] (van den Berg et al.,2006, BMC Genomics 7:142) in the statistical analysis. Mathematically,range-scaling means that every element of column i is divided by therange of column i. The range of column i is the difference between themaximum and minimum value of all elements of column i. Subsequently, thescaled dataset is mean-centered. Mathematically, mean-centering meansthat adding up the values of the individual elements of column i of adata matrix results in zero for column i. This is achieved bysubtracting the mean of column from all values of the individualelements of column i.

PLS Analysis

PLS analysis (Geladi and Kowalski, 1986) was performed in the Matlabenvironment using the PLS Toolbox (version 3.5.4, 2006; EigenvectorResearch, Manson, Wash.).

It is possible that transcripts that show a lot of variation disturb theperformance of the PLS model. Therefore, the model was optimized using ajack-knife approach. A model was build leaving out 10% of the samples,which was repeated until all samples were left out once. For eachtranscript, the relative standard deviation (RSD) was calculated andtranscripts which had an RSD>50% were left out to build a second PLSmodel.

Validation of the Results

A double cross-validation procedure was used to validate the PLS model.In short, two loops are defined: an inner loop and an outer loop. In theinnerloop 90% of the data is used to find an optimal PLS model. In theouterloop a prediction is made for the remaining 10% of the data basedon the innerloop PLS model. This is repeated until all samples are leftout once (so in this case 10 times). Goal of the DCV is to get anindependent estimate of the prediction error. The R² was calculated todetermine how good the original productivity or titer was predicted bythe model. The closer the R² gets to 1, the better the model.

Sequence Analysis of Spots Selected after Transcriptomics Approach

The relevant clones were selected from the glycerol stocks of theordered libraries (gDNA and cDNA library respectively) and cultivated in96-well microtiter plates. The sequences of the inserts from both the 3′and the 5′ end were determined by High Throuput (HT) sequencing service.

All RNA samples were labelled with Cy5. Hybridisations were performedwith all 30 RNA samples, using Cy3-labeled chromosomal DNA of A. terreusas the reference.

The raw transcriptomics data were shown to be of high quality, based onvisual inspection of the arrays after fluorescence scanning. Notably,also the hybridization with the partially degraded RNA samples gave goodresults.

The normalized data were subsequently combined. As the A. terreus arrayconsisted out of two slides, different strategies of combining the datafrom the two slides were pursued, making use of the fact that the cDNAclones are present on both the A and B slide:

-   -   SET 1=mean expression signal of the cDNA clones on slide A and        B, take only those spots that give a signal on both the A and B        slide    -   SET 2=use only the signal of the cDNA spots on the A slide.        Spots with a Signal/Background below 1.5 were removed.    -   SET 3=use only the signal of the cDNA spots on the B slide.        Spots with a Signal/Background below 1.5 were removed.    -   SET 4=Combimean cDNA data of both the A and B slide;        -   i. If both measurement values were zero the combined value            was zero;        -   ii. If both measurements values were both non-zero, the            combined value was equal to the average of the two            measurement values;        -   iii. If one of the two measurement values was zero and the            other measurement value was non-zero, the combined value was            equal to the non-zero measurement value.    -   SET 5=SET 1+normalized gDNA spots using the normalization factor        calculated based on the cDNA clones.

The most relevant spots were subsequently identified by multivariateregression analysis, Multivariate data analysis (MVDA) tools seem verywell suited to prioritize leads from functional genomics datasets. Thesetools take into account the inherent interdependency of biomolecules.These tools allow the identification of the specific genes that are themost important for a specific phenotype by determining the strength ofthe correlation of the expression of every gene with the biologicalquestion under study. Especially the regression tool partial leastsquares (PLS) holds great promise. Principally, the application of PLSresults in a model (equation) that predicts a quantifiable phenotype ofinterest (e.g. itaconic acid titer, P) in terms of the transcripts (A,B, C, . . . ) expressed in A. terreus;P=b ₁ A+b ₂ B+b ₃ C+ . . . .

By subsequently ordering the transcripts based on the absolute value ofthe regression values (i.e. b₁, b₂, b₃, . . . ) transcripts areidentified that contribute the most to itaconate production.

PLS models with both the itaconate titers of the different samples andthe itaconate productivity of the different samples were built usingthese different combined data sets. Moreover, not only linear models butalso logarithmic models were built using the log of the itaconate titer(Table 4). Of these, only the (linear) itaconate titer models werereasonably good models as judged by the (double) cross validationresults and the stability of these models (i.e. R² double crossvalidation≈0.4-0.5)(Table 4). The results of the other PLS models werevery poor, and these models were not pursued.

TABLE 4 Overview of the PLS models build using the transcriptomics dataset X Y R² double cross (data set) (Phenotype) validation PLS model SET1 Productivity 0.10 Titer 0.51 Log(titer) 0.05 SET 2 Productivity 0.13Titer 0.41 Log(titer) 0.18 SET 3 Productivity 0.08 Titer 0.45 Log(titer)0.06 SET 4 Productivity 0.07 Titer 0.40 Log(titer) 0.12 SET 5Productivity 0.08 Titer 0.38 Log(titer) 0.05

Moreover, a second PLS model was built with the titer as the Y-variable,using only the transcripts whose regression value had an RSD<50% (asdetermined based on jack-knifing) in the first PLS model. This were, ingeneral, only some 10-20% of the spots of the complete data set.

‘Top 20’-ies of the combined data data sets analyzed by PLS using theitaconic acid titer as the phenotype were generated. These ‘top-20’-ieswere combined, and unique spots were identified (Table 5 and 6). Intotal 102 of the most relevant spots obtained after PLS analyses (basedon 10 models; 5 data sets, 2 PLS models per data set) were selected forsequencing.

Of the selected spots, >92% were spots belonging to cDNA clones.However, also four of the 5 combined data sets contained only cDNAclones (see above).

Following sequence analysis of the 102 selected spots, the genes presenton these inserts were identified by performing a homology search usingBLAST based on the draft version of the A. terreus genome sequence asavailable from the BROAD institute(http://www.broad.mit.edu/annotation/fgi/).

As different combined data sets/‘top 20’-ies were generated, the overallranking of the transcripts, as reported in Table 5 was based on:

-   -   (i) The frequency of occurrence of an individual spot in the top        20 of the different combined data sets analyzed    -   (ii) The sum of the overall rank of a spot in the different ‘top        20’-ies of the different combined data sets.

Table 5 shows the results of the genes identified on the 20 highestoverall ranking spots identified by PLS analysis based on titer.

TABLE 5 Overall Top 20 PLS analysis - itaconic acid titer. Rank Clone IDGene locus Gene name 1 AsTeR010H08 2 AsTeR037H12 3 AsTeR053F06 4AsTeR037C07 5 AsTeR007H08 6 AsTeR048G12 7 AsTeR023B07 8 AsTeR033F08 9AsTeR035E10 10 AsTeR033A04 11 AsTeR029F07 12 AsTeR045A05 13 AsTeR010H1214 AsTeR041C10 15 AsTeR030D01 ATEG_09972.1 Predicted protein 16AsTeR009D10 17 AsTeR032E02 18 AsTeR032H04 19 AsTeR004H04 20 AsTeR054B06

Moreover, a ranked list of the genes that were identified with thehighest frequency amongst the in total 190 clones sequenced (i.e. 102spots selected after PLS analyses and 88 spots obtained after thedifferential analyses—See EP 08151584)(Table 6).

TABLE 6 Most frequently identified genes amongst the 190 clonessequenced # clones # clones Regulation under from from itaconatedifferential PLS producing Freq. Gene locus approach approach conditionsGene name 25 ATEG_09970.1 25 Up mitochondrial tricarboxylic acidtransporter - EP08151584 19 15 ATEG_09971.1 14 1 Up cis-aconitatedecarboxylase (EP 07112895) 14 11 7 7 4 3 3 2 ATEG_09972.1 2 UpPredicted protein 2 2 2 2 2 2 2 2 The ATEG_00972.1 gene, flankingcis-aconitate decarboxylase (ATEG_09971.1 - EP 07112895), and theputative mitochondrial tricarboxylate transporter protein(ATEG_09970.1 - EP08151584) were identified on two of the spotsidentified by PLS analysis (Table 6) and had an overall rank of 15 inthe combined top 20 (Table 5). Just as the CAD gene and the MTAT gene,the ATEG_09972.1 gene, was found to be upregulated under itaconic acidproducing environmental conditions.

Example 4 Homology Analysis of the ATEG_(—)09972.1 Gene

A BLAST search was performed in order to identify homologous to thepredicted protein ATEG_(—)09972.1 (Table 7). High homologies were onlyfound with genes from two other A. terreus strains. With othermicro-organisms and more specifically fungi, homologues were foundalthough with a relatively low homology. Based on the annotation ofthese homologous genes and the major facilitator superfamily domainidentified in this gene, ATEG_(—)09972.1 was identified as an itaconateexporter.

TABLE 7 BLAST search results with ATEG_09972.1 Identity/ Rank ProteinBest Hit E value Similarity 1 Predicted protein XP_001209274.1 0.0100%/100% A. terreus 2 unknown AAD34564.1 0.0 92%/93% A. terreus 3Unnamed protein BAE57135.1 2e−160 70%/84% product A. oryzae 4 putitativeMFS XP_749665.1 5e−135 64%/77% transporter A. fumigatus 5 putitative MFSXP_001260160.1 5e−134 65%/79% transporter N. fischeri 6 Hypotheticalprotein XP_680781.1 5e−123 59%/74% AN7512.2 A. nidulans 7 Hypotheticalprotein XP_663623.1 1e−105 52%/68% AN6019.2 A. nidulans 8 Unnamedprotein BAE61268.1 1e−91  45%/61% product A. oryzae 9 pH-responsiveXP_001215904.1 2e−91  68%/81% protein 2 precursor A. terreus 10hypothetical XP_383000.1 1e−81  47%/63% protein Fgo2824.1 G. zeae

It appears that at least the gene coding for the cis-aconitatedecarboxylase (ATEG_(—)09971.1) and the gene encoding the mitochondrialtricarboxylate transporter (ATEG_(—)9970.1) lie in the same cluster inthe A. terreus genome (FIG. 2).

A gene neighbouring CAD, the mitochondrial tricarboxylate transporterand the itaconate exporter is a putative regulator containing azinc-finger domain (ATEG_(—)09969.1). This gene was not identified usingour transcriptomics approach, but considering its localization it isexpected that it is relevant for itaconic acid synthesis FIG. 2 showsthat also the lovastatin pathway genes are located on this cluster,suggesting a link between both pathways which are (mainly) specific forA. terreus.

Example 5 (Co-)Expression of the ATEG_(—)09972.1 Gene in AspergillusNiger

In order to unambiguously establish that the ATEG_(—)09972.1 proteinaids to the increased production of itaconic acid, a naturallynon-itaconic acid producing fungal host was (co-)transformed with theCAD gene or transformed with the CAD and MTT (ATEG_(—)09970.1) gene wereco-transformed with the ATEG_(—)09972.1 (MFS) gene.

Expression of the CAD (ATEG 09971.1) Gene in Aspergillus Niger

A PCR generated copy of the gene encoding the CAD protein (seeEP07112895) was generated. For this purpose two sets of primers weregenerated as shown below. PCR amplification based on A. terreus NRRL1960genomic DNA resulted in the isolation of PCR fragments from which thecomplete coding region of the gene encoding the CAD protein, could beisolated as BspHI-BamHI fragments.

CAD full sequence 1529 bp (SEQ ID NO: 11) ORIGIN         BspHI  cadfor40° C.     5′-ATCGTCATGACCAAGCAATCTG-3′(SEQ ID NO: 7)          BspHI  cadfor53° C.    5′-ATCGTCATGACCAAGCAATCTGCGGACA-3′ (SEQ ID NO: 8) 1ATGACCAAGC AATCTGCGGA CAGCAACGCA AAGTCAGGAG TTACGTCCGA AATATGTCAT 61TGGGCATCCA ACCTGGCCAC TGACGACATC CCTTCGGACG TATTAGAAAG AGCAAAATAC 121CTTATTCTCG ACGGTATTGC ATGTGCCTGG GTTGGTGCAA GAGTGCCTTG GTCAGAGAAG 181TATGTTCAGG CAACGATGAG CTTTGAGCCG CCGGGGGCCT GCAGGGTGAT TGGATATGGA 241CAGgtaaatt ttattcactc tagacggtcc acaaagtata ctgacgatcc ttcgtatagA                          (intron) 301AACTGGGGCC TGTTGCAGCA GCCATGACCA ATTCCGCTTT CATACAGGCT ACGGAGCTTG 361ACGACTACCA CAGCGAAGCC CCCCTACACT CTGCAAGCAT TGTCCTTCCT GCGGTCTTTG 421CAGCAAGTGA GGTCTTAGCC GAGCAGGGCA AAACAATTTC CGGTATAGAT GTTATTCTAG 481CCGCCATTGT GGGGTTTGAA TCTGGCCCAC GGATCGGCAA AGCAATCTAC GGATCGGACC 541TCTTGAACAA CGGCTGGCAT TGTGGAGCTG TGTATGGCGC TCCAGCCGGT GCGCTGGCCA 601CAGGAAAGCT CTTCGGTCTA ACTCCAGACT CCATGGAAGA TGCTCTCGGA ATTGCGTGCA 661CGCAAGCCTG TGGTTTAATG TCGGCGCAAT ACGGAGGCAT GGTAAAGCGT GTGCAACACG 721GATTCGCAGC GCGTAATGGT CTTCTTGGGG GACTGTTGGC CCATGGTGGG TACGAGGCAA 781TGAAAGGTGT CCTGGAGAGA TCTTACGGCG GTTTCCTCAA GATGTTCACC AAGGGCAACG 841GCAGAGAGCC TCCCTACAAA GAGGAGGAAG TGGTGGCTGG TCTCGGTTCA TTCTGGCATA 901CCTTTACTAT TCGCATCAAG CTCTATGCCT GCTGCGGACT TGTCCATGGT CCAGTCGAGG 961CTATCGAAAA CCTTCAGGGG AGATACCCCG AGCTCTTGAA TAGAGCCAAC CTCAGCAACA 1021TTCGCCATGT TCATGTACAG CTTTCAACGG CTTCGAACAG TCACTGTGGA TGGATACCAG 1081AGGAGAGACC CATCAGTTCA ATCGCAGGGC AGATGAGTGT CGCATACATT CTCGCCGTCC 1141AGCTGGTCGA CCAGCAATGT CTTTTGTCCC AGTTTTCTGA GTTTGATGAC AACCTGGAGA 1201GGCCAGAAGT TTGGGATCTG GCCAGGAAGG TTACTTCATC TCAAAGCGAA GAGTTTGATC 1261AAGACGGCAA CTGTCTCAGT GCGGGTCGCG TGAGGATTGA GTTCAACGAT GGTTCTTCTA 1321TTACGGAAAG TGTCGAGAAG CCTCTTGGTG TCAAAGAGCC CATGCCAAAC GAACGGATTC 1381TCCACAAATA CCGAACCCTT GCTGGTAGCG TGACGGACGA ATCCCGGGTG AAAGAGATTG 1441AGGATCTTGT CCTCGGCCTG GACAGGCTCA CCGACATTAG CCCATTGCTG GAGCTGCTGA 1501ATTGCCCCGT AAAATCGCCA CTGGTATAA                     cadrev42°C.     BamHI               3′-TTTAGCGGTGACCATATTCCTAGGCCCT-5′(SEQ ID NO: 9)                    cadrev52° C.      BamHI         3′-GGCA

TTTAGCGGTGACCATATTCCTAGGCCCC-5′ (SEQ ID NO: 10)Translation of CAD encoding gene (SEQ ID NO: 12)Total amino acid number: 490, MW=52710

  1 M T K Q S A D S N A K S G V T S E I C H  21W A S N L A T D D I P S D V L E R A K Y  41L I L D G I A C A W V G A R V P W S E K  61Y V Q A T M S F E P P G A C R V I G Y G  81Q K L G P V A A A M T N S A F I Q A T E 101L D D Y H S E A P L H S A S I V L P A V 121F A A S E V L A E Q G K T I S G I D V I 141L A A I V G F E S G P R I G K A I Y G S 161D L L N N G W H C G A V Y G A P A G A L 181A T G K L F G L T P D S M E D A L G I A 201C T Q A C G L M S A Q Y G G M V K R V Q 221H G F A A R N G L L G G L L A H G G Y E 241A M K G V L E R S Y G G F L K M F T K G 261N G R E P P Y K E E E V V A G L G S F W 281H T F T I R I K L Y A C C G L V H G P V 301E A I E N L Q G R Y P E L L N R A N L S 321N I R H V H V Q L S T A S N S H C G W I 341P E E R P I S S I A G Q M S V A Y I L A 361V Q L V D Q Q C L L S Q F S E F D D N L 381E R P E V W D L A R K V T S S Q S E E F 401D Q D G N C L S A G R V R I E F N D G S 421S I T E S V E K P L G V K E P M P N E R 441I L H K Y R T L A G S V T D E S R V K E 461I E D L V L G L D R L T D I S P L L E L 481 L N C P V K S P L V *

The resulting BspHI-BamHI fragment was cloned into the Aspergillusexpression vector pAN52-4-amdS derived from Aspergillus expressionvector pAN52-4. The Aspergillus expression vector pAN52-4-amdS wasderived by cloning the Aspergillus selection marker amdS into theAspergillus expression vector pAN52-4 (EMBL accession #Z32699).

Subsequently, an Aspergillus niger strain AB1.13 (Mattern, I. E. et al.,1992, Mol. Gen. Genet. 234:332-336) was transformed with the CADexpression vector. AmdS transformants resulting for this experiment werepurified by single colony purification and retested for their AmdS+phenotype.

Co-Expression of the CAD Gene and the ATEG 09970.1 Gene in AspergillusNiger

The ATEG_(—)09970.1 gene (MTT) was synthesized (GeneArt) and cloned intoAspergillus niger expression vector pAN52-5doubleNotI by restrictionenzyme cutting sites of double NotI. The expression vectorpAN52-5doubleNotI was derived by adding an extra NotI site in theAspergillus expression vector pAN52-4 (EMBL accession #Z32699).Moreover, the codons of the clone were optimized for expression in theAspergillus niger strain.

Translation of MTT cds (1-861) (SEQ ID NOS:13-14)

Universal code

Total amino acid number: 286, MW=31503

Max ORF starts at AA pos 1 (may be DNA pos 1) for 286 AA (858 bases),MW=31503

  1 ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCT   1 M  S  I  Q  H  F  R  V  A  L  I  P  F  F  A  A  F  C  L  P  61GTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCA  21 V  F  A  H  P  E  T  L  V  K  V  K  D  A  E  D  Q  L  G  A 121CGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCC  41 R  V  G  Y  I  E  L  D  L  N  S  G  K  I  L  E  S  F  R  P 181GAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCC  61 E  E  R  F  P  M  M  S  T  F  K  V  L  L  C  G  A  V  L  S 241CGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTG  81 R  I  D  A  G  Q  E  Q  L  G  R  R  I  H  Y  S  Q  N  D  L 301GTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTA 101 V  E  Y  S  P  V  T  E  K  H  L  T  D  G  M  T  V  R  E  L 361TGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATC 121 C  S  A  A  I  T  M  S  D  N  T  A  A  N  L  L  L  T  T  I 421GGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT 141 G  G  P  K  E  L  T  A  F  L  H  N  M  G  D  H  V  T  R  L 481GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATG 161 D  R  W  E  P  E  L  N  E  A  I  P  N  D  E  R  D  T  T  M 541CCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCT 181 P  V  A  M  A  T  T  L  R  K  L  L  T  G  E  L  L  T  L  A 601TCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGC 201 S  R  Q  Q  L  I  D  W  M  E  A  D  K  V  A  G  P  L  L  R 661TCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCT 221 S  A  L  P  A  G  W  F  I  A  D  K  S  G  A  G  E  R  G  S 721CGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTAC 241 R  G  I  I  A  A  L  G  P  D  G  K  P  S  R  I  V  V  I  Y 781ACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCC 261 T  T  G  S  Q  A  T  M  D  E  R  N  R  Q  I  A  E  I  G  A 841TCACTGATTAAGCATTGGTAA 281  S  L  I  K  H  W  *

Subsequently, an Aspergillus niger strain AB1.13 (Mattern, I. E. et al.,1992, Mol. Gen. Genet. 234:332-336) was co-transformed with the CADexpression vector and the MTT expression vector. AmdS transformantsresulting for this experiment were purified by single colonypurification and retested for their AmdS+ phenotype.

Co-Expression of the Cad Gene, MTT Gene (ATEG 09970.1) Together with theATEG-09972.1 Gene in Aspergillus Niger

The ATEG-09972.1 (MFS) gene was synthesized (GeneArt) and cloned intoAspergillus niger expression vector pAN52-5doubleNotI by restrictionenzyme cutting sites of double NotI. The expression vectorpAN52-5doubleNotI was derived by adding an extra NotI site in theAspergillus expression vector pAN52-4 (EMBL accession #Z32699).Moreover, codons of the clone were optimized for expression in theAspergillus niger strain.

Translation of MFS cds (1-861) (SEQ ID NOS:15-16)

Universal code

Total amino acid number: 286, MW=31503

Max ORF starts at AA pos 1 (may be DNA pos 1) for 286 AA 9858 bases),MW=31503

  1 ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCT   1 M  S  I  Q  H  F  R  V  A  L  I  P  F  F  A  A  F  C  L  P  61GTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCA  21 V  F  A  H  P  E  T  L  V  K  V  K  D  A  E  D  Q  L  G  A 121CGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCC  41 R  V  G  Y  I  E  L  D  L  N  S  G  K  I  L  E  S  F  R  P 181GAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCC  61 E  E  R  F  P  M  M  S  T  F  K  V  L  L  C  G  A  V  L  S 241CGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTG  81 R  I  D  A  G  Q  E  Q  L  G  R  R  I  H  Y  S  Q  N  D  L 301GTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTA 101 V  E  Y  S  P  V  T  E  K  H  L  T  D  G  M  T  V  R  E  L 361TGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATC 121 C  S  A  A  I  T  M  S  D  N  T  A  A  N  L  L  L  T  T  I 421GGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT 141 G  G  P  K  E  L  T  A  F  L  H  N  M  G  D  H  V  T  R  L 481GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATG 161 D  R  W  E  P  E  L  N  E  A  I  P  N  D  E  R  D  T  T  M 541CCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCT 181 P  V  A  M  A  T  T  L  R  K  L  L  T  G  E  L  L  T  L  A 601TCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGC 201 S  R  Q  Q  L  I  D  W  M  E  A  D  K  V  A  G  P  L  L  R 661TCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCT 221 S  A  L  P  A  G  W  F  I  A  D  K  S  G  A  G  E  R  G  S 721CGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTAC 241 R  G  I  I  A  A  L  G  P  D  G  K  P  S  R  I  V  V  I  Y 781ACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCC 261 T  T  G  S  Q  A  T  M  D  E  R  N  R  Q  I  A  E  I  G  A 841TCACTGATTAAGCATTGGTAA 281  S  L  I  K  H  W  *

Subsequently, an Aspergillus niger strain AB1.13 (Mattern, I. E. et al.,1992, Mol. Gen. Genet. 234:332-336) was co-transformed with the CADexpression vector, the MTT expression vector and the MFS expressionvector. AmdS transformants resulting for this experiment were purifiedby single colony purification and retested for their AmdS+ phenotype.

Analysis of A. Niger Transformants for Itaconic Acid Production

Several positive transformants and the parental host strain weresubsequently cultured in Shake Flask in MM medium supplied with uridinecontaining glucose as C-source and nitrate as N-source. Medium samplesfrom the various cultures were analyzed by HPLC for the presence ofitaconic acid (Table 8).

Shake Flask Medium Compositions:

Per liter: 0.52 g of KCl, 2.4 g of NaNO₃, 1.56 g of KH₂PO₄, 0.24 g ofMgSO₄*7H₂O, 5 mg of Fe(III) SO₄*7H₂O, 5 mg of MnCl₂*4H₂O, 0.022 g ofZnSO₄*7H₂O, 0.011 g of H₃BO₃, 1.7 mg of CoCl₂*6H₂O and 2.44 g ofuridine, 100 g of glucose as a carbon source. All media were prepared indemineralised water.

HPLC analysis was performed with a reversed phase column, using aDevelosil™ 3 μm R^(P)-Aqueous C30 140A column at a constant temperatureof 25° C., with elution with 20 mM NaH2PO4, pH 2.25 and acetonitril.Compounds were detected by UV at 210 nm using a Waters 2487 Dualwavelength Absorbance detector (Milford, Mass., USA). Retention time ofitaconic acid was 18.82 min.

TABLE 8 Itaconic acid concentration in the culture fluid of the A. nigerAB1.13 transformants cultivated in shake flasks. Aspergillus niger AB1.13 transformants (AB 1.13 CAD) itaconic acid strain code time (hrs)mg/g wet weight AB 1.13 WT 54 0 AB 1.13 CAD 5.1 54 1.0 AB 1.13 CAD 7.254 0.7 AB 1.13 CAD 10.1 54 1.4 AB 1.13 CAD 14.2 54 1.2 AB 1.13 CAD 16.154 1.2 AB 1.13 CAD + MTT 4.1 54 1.3 AB 1.13 CAD + MTT 6.2 54 1.5 AB 1.13CAD + MTT 2.2.1  54 2.2 AB 1.13 CAD + MTT + MFS 9.2.1* 54 2.3 AB 1.13CAD + MTT + MFS 9.2.2* 54 2.5 AB 1.13 CAD + MTT + MFS 9.4.1* 54 2.5 AB1.13 CAD + MTT + MFS 12.1.1  54 2.3 *Duplicate isolates of the sametransformant.

No itaconic acid was detected in the supernatant of the parental strainwhile in the culture fluid of the strains containing the CAD gene(strains marked CAD), itaconic acid was detected.

In both the culture fluid of the strains containing the CAD gene and thestrains containing both the CAD gene and MTT gene (strains markedCAD+MTT) and the strains containing the CAD, the MTT and the MFS gene(strains marked CAD+MTT+MFS), itaconic acid was detected (Table 8). Inall MFS expressing strains more itaconic acid was produced in theculture fluid than in the strains expressing only the CAD gene, or theCAD and the MTT gene. Moreover, the average itaconic acid concentrationwas higher in the culture fluid of the strains expressing the MFS, theCAD and the MTT gene compared to the transformants expressing only theCAD or the CAD+MTT gene: 2.4 mg itaconic acid/g mycelial wet weightversus 1.1 mg itaconic acid/g mycelial wet weight (CAD gene only) and1.7 mg itaconic acid/g mycelial wet weight (CAD+MTT gene—Table 8).

The invention claimed is:
 1. A method for enhancing the production ofitaconic acid in a micro-organism that is capable of producing itaconicacid said method comprising increasing the activity of an itaconatetransporter protein in said microorganism, wherein the increasing iseffected by expressing a nucleotide sequence encoding said itaconatetransporter protein wherein the itaconate transporter protein has theamino acid sequence of SEQ ID NO:3 or an amino acid sequence at least95% identical thereto or SEQ ID NO:6, and culturing said microorganismunder conditions whereby itaconic acid is produced.
 2. The method ofclaim 1, wherein the nucleotide sequence encodes a protein having SEQ IDNO:3 or SEQ ID NO:6.
 3. The method of claim 1, wherein themicro-organism is a citrate producing micro-organism.
 4. The method ofclaim 1, wherein the micro-organism is a lovastatin producingmicro-organism.
 5. The method of claim 1, wherein the expressing iseffected by providing a vector comprising a promoter operably linked tosaid nucleotide sequence.
 6. The method of claim 1, further comprisingisolating itaconic acid from the extracellular medium.
 7. The method ofclaim 3 wherein the citrate producing microorganism is A. terreus, A.niger, A. itaconicus, A. nidulans, A. oryzae, A. fumigates, Yarrowialipolytica, Ustilago zeae, Candida sp., Rhodotorula sp., Pseudozymaantarctica, E. coli, or Saccharomyces cerevisiae.
 8. The method of claim4 wherein the lovastatin producing microorganism is Monascus spp.,Penicillium spp., Hypomyces spp., Doratomyces spp., Phoma spp.,Eupenicillium spp., Gymnoascus spp., Pichia labacensis, Candidacariosilognicola, Paecilomyces variotii, Scopulariopsis brevicaulis orTrichoderma spp.
 9. The method of claim 1 wherein the nucleotidesequence comprises SEQ ID NO:2 or a nucleotide sequence at least 95%identical thereto or SEQ ID NO:5.
 10. The method of claim 1 wherein thenucleotide sequence comprises SEQ ID NO:2 or SEQ ID NO:5.